This administrative supplement request through the Office of Dietary Supplements (PA-16-319) takes advantage of the expertise of one member of the multi-PI group of scientists on the Parent Award ?Phenotyping embryonic lethal knockout mice with neural crest and neural tube defects?. The Niswander lab has extensive experience in the detailed characterization of embryonic mutations that disrupt neural tube closure leading to neural tube defects (NTDs) like spina bifida. NTDs are a common birth defects (1 in 1000 births) and the mouse represents an excellent model of human neural tube closure and has led to the discovery of numerous genes that control neural tube development and that are now associated with human NTDs. The original goal in Aim 2 under Dr. Niswander?s direction is to delve deeply into the cellular and molecular mechanisms responsible for defects in neural tube formation in mice arising from the Knock-Out Mouse Project (KOMP2) to better understand the genetic networks that orchestrate neural tube closure. Folate deficiency has long been associated with increased risk for NTD-affected pregnancies. Recommendations to increase folate levels in women of child-bearing age and subsequent fortification of flour with folic acid have decreased the incidence of NTDs by nearly 30%. Yet, still very little is known as to how folic acid acts to decrease the NTD incidence. Moreover, the few studies in mice suggest that there is a range of responses to folic acid fortification, depending on the genetic risk factors, from beneficial to no response to detrimental ? highlighting the importance of understanding which genetic variants and perhaps molecular pathways are favorably influenced by folic acid fortification. Furthermore, despite the decades-long folic acid fortification campaign, the possible consequences of long-term and potential epigenetic changes on NTD risks and mechanisms have not been studied. The request for an administrative supplement will allow these outstanding questions to be addressed. The requested experiments take advantage of two novel mutant mouse lines characterized in the parent grant, one that results in spina bifida and the other in cranial NTD, in conjunction with mouse lines that show altered NTD risk dependent on the dose and duration of FA exposure. Within two new Aims, the goals here are to gain insight into the phenotypic and molecular changes induced by folic acid fortification over multiple generations in NTD models. The ultimate goal is to discover expression biomarkers that may help guide individualized recommendations for optimal outcomes.